NecroPT - Targeting Hepatocyte Necroptosis to Treat Liver Diseases

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Design and synthesis of small molecule modulators of p53

Publication . Ribeiro, Carlos Jorge Azevedo Costa, 1980-; Santos, Maria Manuel Duque Vieira Marques dos; Moreira, Rui, 1960-; Rodrigues, Cecília M. P., 1968-

Among the tumor suppressor genes, p53 is one of the most studied. It is widely regarded as the “guardian of the genome”, playing a pivotal part in the preservation of genomic integrity by regulating cell cycle, apoptosis, DNA repair, senescence and angiogenesis, and consequently has a major role in carcinogenesis. The function played by p53 in tumor suppression is further highlighted by the fact that direct inactivation of this gene occurs in more than 50% of malignancies. In addition, in tumors that retain wild type p53 status, its function is usually inactivated by overexpression of negative regulators, primarily murine double minute-2 (MDM2), mainly through MDM2 gene amplification or by activity loss of MDM2 inhibitor ARF. Hence, restoring p53 function in cancer cells represents a valuable anticancer approach. Several strategies are being developed, and in particular targeting p53-MDM2 interaction has emerged as a promising viable approach when dealing with cancers that retain wild type p53 function. These two proteins regulate each other through an autoregulatory feedback loop: activation of p53 stimulates the transcription of MDM2, which in turn binds to the Nterminal transactivation domain of p53, disabling its transcriptional function. p53- MDM2 interaction inhibitors share common structural features: a rigid heterocyclic scaffold with three lipophilic groups that mimic the three pivotal p53 Phe19, Trp23 and Leu26 that interact with MDM2. Seven compounds have already entered clinical trials. The main goal of this PhD thesis was to develop new anticancer agents containing a spirooxindole scaffold with different spiro five-membered rings: isoxazoline, oxadiazoline, and triazolines. The spirocycle can potentially function as the rigid heterocyclic scaffold, from which three lipophilic groups are projected to mimic the three pivotal p53 amino acids. This work followed three major strategies: synthesis of spirooxindole derivatives by 1,3 dipolar cycloaddition; biological evaluation of the compounds synthesized; and stability assessment. Overall this PhD thesis contributed with three new families of spirooxindoles with in vitro anti-cancer activity. The most active derivative possessed a GI50 of 1.72 μM in HCT116 p53(+/+) cell line. Furthermore their ability to disrupt the interaction between p53 and MDM2 was confirmed by implementing a cell-base in vitro bimolecular fluorescence complementation assay (BiFC) and the apoptotic outcome verified by immunoblotting analysis and luminescent caspase 3/7 activity assay.

2015Doctoral thesis

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